The serum albumins belong to a multigene family of proteins that includes alpha-fetoprotein and human group-specific component, also known as vitamin-D binding protein. The members of this multigene family are typically comprised of relatively large multi-domain proteins, and the serum albumins are the major soluble proteins of the circulatory system and contribute to many vital physiological processes. Serum albumin generally comprises about 50% of the total blood component by dry weight, and as such is responsible for roughly 80% of the maintenance of colloid osmotic blood pressure and is chiefly responsible for controlling the physiological pH of blood.
The albumins and their related blood proteins also play an extremely important role in the transport, distribution and metabolism of many endogenous and exogenous ligands in the human body, including a variety of chemically diverse molecules including fatty acids, amino acids, steroids, calcium, metals such as copper and zinc, and various pharmaceutical agents. The albumin family of molecules are generally thought to facilitate transfer many of these ligands across organ-circulatory interfaces such as the liver, intestines, kidneys and the brain, and studies have suggested the existence of an albumin cell surface receptor. See, e.g., Schnitzer et al., P.N.A.S. 85:6773 (1988). The albumins are thus intimately involved in a wide range of circulatory and metabolic functions.
Human serum albumin (HSA) is a protein of about 66,500 kD and is comprised of 585 amino acids including at least 17 disulphide bridges. As with many of the members of the albumin family, human serum albumin plays an extremely important role in human physiology and is located in virtually every human tissue and bodily secretion. As indicated above, HSA has an outstanding ability to bind and transport a wide spectrum of ligands throughout the circulatory system including the long-chain fatty acids which are otherwise insoluble in circulating plasma. The atomic structure and particular details regarding the binding affinities of albumin and the specific regions primarily responsible for those binding properties have been previously determined as set forth, e.g., in U.S. patent application Ser. No. 08/448,196, filed May 25, 1993, now U.S. Pat. No. 5,780,594 and U.S. patent application Ser. No. 08/984,176, filed Dec. 3, 1997, now U.S. Pat. No. 5,948,609, both of which are incorporated herein by reference.
In addition to human serum albumin, studies have been made on albumins in a variety of animal species, and it has been determined that over 60% of the amino acid sequences are conserved among the known albumin sequences of many mammals such as bovine, rat and human serum albumin. Moreover, as more and more albumins from other animal species have been sequenced, it has been found that the albumins from a wide range of vertebrate species including sheep, frogs, salmon, mice, pigs and even sea lampreys share varying degrees of sequence homology, and all share the characteristic repeating pattern of disulphide bridges observed in human serum albumin, thus implying a common three-dimensional structure. Furthermore, all members of the albumin multigene family for which sequences have been determined appear to have internal sequence homology (from two- to seven-fold), thus suggesting that the proteins evolved from a common ancestral protein, and reflecting the vital nature and function of this protein. See, e.g., Carter et al., Science 244:1195 (1989).
Because of the vital role played by albumins, there are literally thousands of applications for serum albumin and its related proteins covering a wide range of physiological conditions, and most often, native serum albumin has been used. However, unlike blood proteins such as hemoglobin, native serum albumins are non-functional as oxygen transport systems, and thus have not been useful in blood replacement systems requiring oxygen transport. More recently, an oxygen-transporting albumin-based blood replacement composition was developed which can be utilized as a blood volume expander, as has been disclosed in U.S. Pat. No. 5,948,609, incorporated herein by reference, and this composition further increases the importance and usefulness of serum albumin.
Additionally, in applications involving albumin, it has been known to utilize the human serum albumin sequence of the prototypical or major allotype of the human serum albumin sequence, which has included the well known n-terminal amino acid sequence n-DAHK-c. See, e.g., Carter et al., Advances in Protein Chemistry, Vol. 45, 153–203 (1994) and Peters, “All About Albumin”, Academic Press (1995). The binding of copper and nickel to the n-terminal peptide of albumins has been known and studied for many years, and this site has been designated as functional binding location Site VI in Carter et al. (1994). The sequence X—X-Histidine appears to be the key to the copper and nickel metal binding at this site, and of additional importance is the structural flexibility of the n-terminal polypeptide which cannot be structurally hindered.
At physiological pH, copper is bound with extraordinarily high affinity to this site (Ka=1.6×1016 M−1) (see Camerman et al., Can. J. Chem., Vol. 54:1309–1316 (1976)), perhaps the highest reported binding constant of any bound ligand to serum albumin. By comparison, the binding for Nickel is Ka=3×105 M−1. See Lau et al., J. Biol. Chem. Vol. 249:5878–5884 (1974). While this feature of the albumin molecule serves to protect the body from the potential damaging influences of the metals, especially copper, the nickel complex with albumin is known to elicit allergic reactions in some individuals, which occurs following ingestion of Ni(II) or exposure to nickel plated jewelry or other similar items. For example, an occupational asthma resulting from nickel binding is well recognized and has been traced to antibodies against Ni(II) specifically bound to the n-terminus of human serum albumin. See Carter et al. (1994), supra and Nieboer et al., Br. J. Ind. Med., Vol. 41:56–63 (1984).
In the normal course of recombinant production of albumin and other proteins, there is usually a given level of certain metals, including nickel and copper, which are required as components of the culture media and used in albumin production. Consequently, a significant level of nickel, copper and/or other metals is chelated by the n-terminal peptide of albumin during production, as evidenced during production by the green and yellow coloration of the recombinant human serum albumin. However, the presence of these metals, even in trace amounts, in the albumin produced via recombinant methods can lead to significant health problems as indicated above.
There is thus a significant need to develop safe and effective serum albumin products for use in many applications, particularly those which involve use in humans either internally or externally, which can reduce or eliminate the high affinity of albumin to copper and nickel and/or other metals, and which can thus reduce the risk that a potential albumin-based product will elicit an allergic response to the bound metal in a human or animal who is being treated with an albumin composition.